PepGen P-15是最近研發的人工合成異質移植骨材，是由無機礦物質 anorganic bovine mineral materials (ABM)加上一段人工合成第一型膠原纖維α鏈15個胜肽-GTPGPQGIAGQRGVV-(P-15)，融合而成的異質移植骨材。其中無機礦物質組成不僅僅是扮演了一個載體的角色，負責將15個膠原蛋白胜肽鏈呈現在作用的細胞以及接觸的細胞外間質，並且在礦物質生成方面具有骨質生成傳導的作用。在有機質方面，15個胜肽短鏈已經被證實與膠原蛋白的生成及結構決定有相當重要的關連。而且有報告指出15個胜肽短鏈與造骨母細胞結合作用後，可能能夠使其增生和促進分化並且增加細胞的遷移及聚集。之前已有許多學者進行在體外細胞間以及牙周組織缺損病灶的填補治療，PepGen P-15都有不錯的骨生成作用。但是對於PepGen P-15對人類牙周韌帶纖維母細胞的作用機制並不清楚，因此我們進行這研究的目的則是希望藉由PepGen P-15對人類牙周韌帶纖維母細胞的作用來觀察，並藉由生物化學的分析檢驗方法，來探討PepGen P-15對人類牙周韌帶纖維母細胞的作用機轉，以及這種骨移植材料對細胞的生化作用影響，包括生成、分化、鹼性磷酸酶活性及礦物質化能力的影響。我們的蛋白質質體鑑定分析實驗結果顯示，牙齦纖維母細胞上的annexin II蛋白可與這種骨移植材料結合，另一種單純無機礦物質Osteograf N-300則明顯較少結合。經由西方點墨法證實annexin II可存在於細胞膜，因此活體狀況下有機會與PepGen P-15直接作用。兩種骨移植材料對細胞的分裂、分化、鹼性磷酸酶活性及礦物質化有所差異，PepGen P-15明顯優於Osteograf N-300。未來我們需要更直接的證據，例如siRNA或者胜肽競爭才可以推論纖維母細胞上的annexin II蛋白確實可以和PepGen P-15結合，進而直接影響纖維母細胞在細胞增生和礦物質化有較好的促進效果。

　　PepGen P-15 is a recently developed bone replacement graft. The graft consists of anorganic bone mineral (ABM) particle coating with a putative cell-bonding domain of type-I collagen (P-15 peptide) to mimic the bone matrix compound. The amino acids are synthetic replications of residues 766-780 of the α chain of type I collagen, which plays important roles in cell binding. In previous vivo and vitro studies, it has been demonstrated that the attachment of human fibroblast to hydroxyapatide particles is increased when p-15 is present on the particles. In this study, we tried to explore the mechanism of how fibroblasts bind to PepGen p-15 and investigated the downstream biological functions of the PepGen p-15 on the cultured human periodontal ligament fibroblasts.

　　Annexin II is a protein involving in osteoblastic mineralization, and when overexpressed, it led to an increase in alkaline phosphatase activity as well as mineralization. By biochemical analysis, we found that annexin II and vimentin protein can bind to PepGen P-15. We also demonstrated that annexin II exists on cell membrane and cytoplasm. Annexin II on cell membrane has chance to encounter PepGen P-15 and effect the signal transduction to facilitate cell growth and differentiation, such as alkaline phosphatase activity and mineralization. According to our result, annexinⅡ can bind to the peptide of a bio-mimetic bone graft and may influence its biological effect. PepGen P-15 can enhance the growth and mineralization of PDL fibroblast. In the future, we will employ SiRNA and/or competitive amino acids to interfere the binding and observe the influenced consequence. This may provide more direct evidence that annexin II regulates the biological functions of PDL fibroblast.

1.Balcerzak M., Hamade E., Zhang L., Pikula S., Azzar G., Radisson J., Bandorowicz-Pikula J., and Buchet R. The roles of annexins and alkaline phosphatase in mineralization process. Acta Biochimica Polonica , 50:1019-1038, 2003

2.Baran D.T., Quail J.M., Ray R., Leszyk J., and Honeyman T. Annexin II is the membrane receptor that mediates the rapid actions of 1α,25-Dihydroxyvitamin D3. Journal of Cellular Biochemistry, 78: 34-46, 2000

3.Bhatnagar R.S., Qian J.J., and Gough C.A. The role in cell binding of a β-bend within the triple helical region in collagen α1(1) chain : structural and biological evidence for conformational tautomerism on fiber surface. Journal of Bimolecular Structure & Dynamics ,14 : 547-560, 1997

4.Bhatnagar R.S., Qian J. J., Wedrychowska A., Sadeghi M., Wu Y. M., and Smith N. Design of biomimetic habitats for tissue engineering with P-15, a synthetic peptide analogus of collagen. Tissue Engineering, 5: 53-65, 1999

5.Bonewald L.F., Schwartz Z., Swain L.D., Ramirez V., Poser J., Boyan B.D., Stimulated of plasma membrane and matrix vesicle enzyme activity by transforming growth factor-beta in osteosarcoma cell culture. Journal of Cellular Physiology, 145: 200-206, 1990

6.Bonson S., Jeansonne B.G., and Lallier T.E. Root-end filling materials alter fibroblast differentiation. Journal of Dental Research, 83: 408, 2004

7.Carinci F., Pezzetti F., Volinia S., Laino G., Arcelli D., Caraelli E., Degidi M., and Piattelli A. P-15 cell-binding domain derived from collagen: analysis of MG63 osteoblastic -cell response by means of a microarray technology. Journal of Periodontology, 75 : 66-83, 2004

8.Degidi M., Piattelli M., Scarano A., Iezzi G., and Piattelli A. Bone replacement graft material enhanced by synthetic peptide (P-15). Journal of Oral Implantology, 30 : 376-383, 2004

9.Gerke V., and Moss S.E. Annexin II: from structure to function. Physiological Reviews , 82: 331-371 , 2001

10.Gillette J.M., Chan D.C., Nielsen-Preiss S.M. Annexin 2 expression is reduced in human osteosarcoma metastasis. Journal of Cellular Biochemistry, 92:820-832, 2004

11.Gillette J.M., and Nielsen-Preiss S.M. The role of annexin 2 in osteoblastic mineralization. Journal of Cell Science, 117: 441-449, 2003

12.Groeneveld M.C., Everts V., and Beertsen W. Alkaline phosphatase activity in the periodontal ligament and gingival of the rat molar: its relation to cementum formation. Journal of Dental Research, 74 : 1374-1381, 1995
13.Helfand B.T., Chang L., and Goldman R.D. Intermediate filaments are dynamic and motile elements of cellular architecture , Journal of Cell Science, 117: 133-141 , 2004

14.Kleinman H.K., Klebe R.J., and Martin G.R. Role of collagenous matrices in the adhesion and growth of cells. Journal of Cell Biology, 88 : 473-485, 1981

15.Koru L., Parker M.H., Griffiths G.S., Newman H.N., Olsen I. Flow cytometry analysis of gingival and periodontal ligament cells. Journal of Dental Research, 77: 555-564, 1998

16.Kubler A., Neugebauer J., oh J.H., Scheer M., and Zoller J.E. Growth and proliferation of human osteoblast on different bone graft substitutes: an in vitro study. Implant Dentistry, 13:171-179, 2004

17.Lallier T.E., Yukna R., Marie S.S., and Moses R. The putative collagen binding peptide hastens periodontal ligament cell attachment to bone replacement graft material. Journal of Periodontology, 72 : 990-997, 2001

18.Lambert O., Cavusoglu N., Gallay J., Vincent M., Rigaud J.L., Henry J.P., and Ayala-Sanmartin J. Novel organization and properties of annexin 2-membrane complexes. The Journal of Biological Chemistry , 279: 10872–10882, 2004

19.Mornet E., Stura E., Lia-Baldin A., Stigbrand T., Menez A., and Le Du M. Structural evidence for a functional role of human tissue nonspecific alkaline phosphatase in bone mineralization. The Journal of Biological Chemistry , 276: 31171-31178, 2001

20.Nakamura Y., Noda K., Shimpo S., Oikawa T., Kawasaki K., Hirashita A. Phosphatidylinositol-dependent bond between alkaline phosphatase and collagen fibers in the periodontal ligament of the rat molar. Histochemistry and Cell Biology, 121: 39-45, 2004

21.Nguyen H., Qian J.J., Bhatnagar R.S., and Li S. Enhanced cell attachment and osteoblastic activity by P-15 peptide-coated matrix in hydrogels. Biochemical and Biophysical Communications, 311: 179-186, 2003

22.Qian J.J., and Bhatnagar R.S. Enhanced cell attachment to anorganic bone mineral in the presence of a synthetic peptide related to collagen. Journal of Biomedical Materials Research, 31: 545-554, 1996

23.Serre C.M., Papillard M., Chavassieux P., and Boivin G. In vitro induction of a calcifying matrix by biomaterials constituted of collagen and/or hydroxyapatide: an ultrastructural comparison of the three types of biomaterials. Biomaterials, 14: 97-106, 1993

24.Seyedlin S.M., and Alto P., Osteoinduction : a report on the discovery and research of unique protein growth factors mediating bone development. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics, 68 : 527-531, 1989

25.Siever D. A., and Erickson H.P. Extracelluar annexin II. International Journal of Biochemistry and Cell Biology, 29 : 1219-1223, 1997

26.Sogal A., and Tofe A.J. Risk assessment of bovine spongiform encephalopathy transmission through bone graft material derived from bovine bone used for dental applications. Journal of Peridonotology, 70 : 1053-1063, 2004

27.Stanford C.M., Jacobson P.A., Eanes E.D., Lembke L.A., and Midura R.J. Rapidly forming apatitic mineral in an osteoblastic cell line. The Journal of Biological Chemistry, 270: 9420-9428, 1995

28.Srasatti C., Spears R., Gutmann J. L., and Opperman L. A. Increased Tgf-β1 production by rat osteoblasts in the presence of PepGen P-15 in Vitro. Journal of endodontics , 30 : 213-217, 2004

29.Urist M.R., DeLang R.J., and Fineman G.A. Bone cell differentiation and growth factor. Science , 220 : 680-686, 1981

30.Valentin A. H., and Weber J. Receptor technology – cell binding to P-15: a new method of regenerating bone quickly and safely-preliminary histomorphometrical and mechanical results in sinus floor augmentations. Keio J.M., 53: 166-171, 2004

31.Wang N., and Stamenovic D., Mechanics of vimentin intermediate filament. Journal of Muscle Research and Cell Motility, 23: 535-540, 2002

32.White M.P. Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocrine Reviews , 15: 439-461, 1994

33.Yukna R.A., Callan D.P., krauser J.T., Evans G.H., Aichelman-Reidy M.E., Moore K, Cruz R., and Scott J.B. Multi-center clinical evaluation of combination anorganic bovine-derived hydroxyapatide matrix (ABM)/cell binding peptide (P-15) as a bone replacement graft material in human periodontal osseous defects. 6-month results. Journal of Peridonotology , 69: 655-663, 1998

34.Yukna R.A., Krauser J.T., Callan D.P., Evans G.H., Cruz. R. Multi-center clinical comparison of combination anorganic bovine-derived hydroxyapatide matrix (ABM)/cell binding peptide (P-15) and ABM in human periodontal osseous defects. 6-month results. Journal of Peridonotology , 71: 1671-1679, 2000

35.Yukna R. A., Krauser J.T., Callan D.P., Evans G.H., Cruz R, and Martin M. Thirty -six month follow-up of 25 patients treated with combination anorganic bovine-derived hydroxyapatide matrix (ABM)/cell binding peptide (P-15) bone replacement grafts in human infrabony defects. I. Clinical Finding. Journal of Peridonotology , 73: 123-128, 2002

36.吳慧芬、呂麗琪，質譜儀分析技術的突破開展生化科技新領域，科學發展，362：49~51，2003。